Immunogenicity And Protection Efficacy Of An Adenovirus-based Bicistronic Candidate Vaccine Expressing Nsp4 And Vp7 Of Rotavirus

Rotavirus (RV) is the leading cause of virus associated gastroenteritis among infants and children under five years old worldwide. RV can be divided into 7 groups (A-G) according to VP6 antigen specificity. Human infant diarrheas mainly result from group A rotavirus. More than 100 million acute diarrhea episodes,2 million hospitalizations and over half a million deaths attribute to rotavirus infection every year. There is no specific drug available for RV diarrhea therapy. Vaccination is still the preferred means to prevent the RV infection, and decrease the severe and fatal cases.Nowadays, RV programmed immunization has been implemented in various countries and regions. The licensed RV vaccines, RotaTeq, Rotarix and Luotewei (LLR), are live attenuated vaccines. These vaccines contribute greatly to reduce the morbidity and mortality of RV infection. But the efficacies of live vaccines in middle-low income regions are unsatisfactory. It could be multiple reasons like selected RV strain, different geographical, economic condition, and pre-existed antibodies from breast feeding. Furthermore, there are some potential safety problems for live vaccines including foreign factor contamination, occasional adverse reactions like intussusception, and virulent recovery of vaccine virus. Therefore, an alternative strategy for RV vaccine is to develop an inactive or genetic engineering vaccine. Here, the multifunctional nonstructural protein NSP4 and the capsid protein VP7 of rotavirus are chosen as interest proteins to construct an adenovirus-based bicistronic recombinant vaccine. And the immunogenicity and efficacy of this vaccine are evaluated in mice.The construction of recombinant adenoviruses and expressing detection of interested proteins in vitro. In first place, the NSP4 and VP7 genes of G1P[8] type rotavirus ZTR-68 strain were amplified by reverse transcription-polymerase chain reaction (RT-PCR). The obtained VP7 and NSP4 genes were introduced into the multi-cloning site (MCS) A and MCS B of eukaryotic vector pcDNA3.0BA in turn. Subsequently, the DNA fragment carrying NSP4-polyA-CMV-VP7 was amplified by polymerase chain reaction (PCR) using pcDNA3.0-NSP4-VP7 as template, and cloned into the adenovirus shuttle vector pshuttle-CMV. So the recombinant plasmid pshuttle-NSP4-VP7, in which NSP4 and VP7 genes were located separately on the downstream of two CMV promoters, was constructed. Next, the pshuttle-NSP4-VP7 was co-transformed with the adenovirus-backbone plasmid pAdeasy-1 into E, coli BJ5183 using electroporation for homologous recombination. The selected positive recombinant plasmid pAd-NSP4-VP7 was linearized and transfected into the Ad293 packaging cells for generation of infectious recombinant adenoviruses (rAd-NSP4-VP7). At the same time, another recombinant adenoviruses rAd-VP7 and rAd-NSP4, which both carried one single interest gene, were constructed in the similar methods. In result, all three recombinant adenovirus packaged and the virus particles were observed by transmission electron microscope. Furthermore, the expression of interest protein NSP4 and VP7 could be detected by western blot and indirect immunofluorescence asssy.Evaluation of immunogenicity of the recombinant adenoviruses in mice. After recombinant adenovirus’(rAd-NSP4-VP7> rAd-NSP4 and rAd-VP7) passaged serially in Ad293 cells, ICR mice were immunized with them respectively by intramuscular or intranasal route. All three recombinant adenovirus induced antibodies production including sera IgG, IgA and intestinal secretory IgA in different level. Even neutralizing antibodies against RV ZTR-68 strain could be detected in all recombinant adenovirus immunized group. Higher sera neutralizing titers acquired in rAd-NSP4-VP7 and rAd-VP7 immunized group among experimental mice. Furthermore, the percentage of the mice spleen lymphocytes subsets of CD4, CD8, and CD69 positive were detected by flow cytometry (FCM), whereas the secretions of IFN-y and IL-4 by stimulated lymphocytes were analyzed using enzyme-linked immunospot (ELISPOT). It indicated that all the three adenoviruses induced antigen-specific cellular immune responses, of which the rAd-NSP4-VP7 and rAd-NSP4 immunized group appeared more robust.The protective efficacy of rAd-NSP4-VP7 against RV challenge in suckling mice. In addition, BALB/c and ICR suckling mice were inoculated with different RV strains orally to induce the diarrhea or fecal shedding of RV. The animal shedding model of RV Wa and ZTR-68 strains infecting suckling mice was established. Followed by the ICR pregnant mice were immunized with the rAd-NSP4-VP7 vaccine, the shedding rate of new born suckling mice declined significantly after challenge. The protective efficacy of the rAd-NSP4-VP7 vaccine was proved.This study demonstrates the construction of the bicistronic recombinant adenovirus rAd-NSP4-VP7 expressing NSP4 and VP7. The robust immunogenicity of rAd-NSP4-VP7 was confirmed since it elicited systematic immune response including humoral, cellular and mucosal immune response in mice. Furthermore, the rAd-NSP4-VP7 showed protective efficacy for suckling mice shedding against RV challenge. Meanwhile, the immunogenicity comparison between bicistronic rAd-NSP4-VP7 and recombinant adenovirus expressing single interest gene (rAd-NSP4 or rAd-VP7) was made. In conclusion, this study provided a basis for adenovirus-vectored NSP4 and VP7 as both RV vaccine candidates, and explored the development of the new genetic engineering vaccine against rotavirus infection.